Establishing Of A Cell Line For Inhibiting Orf Virus By RNA Ineterference Technology

Orf, also knowed as contagious ecthyma, which is caused by orf virus, is a common epitheliotrophic viraldisease of sheep、goats and wild ruminants. Humans are also susceptible to orf. The disease which has highinfectivity and morbidity is found throughout the world. The eating and swallowing of the sick animal will beseriously affected by its mouth and lip′s lesion. It was reported that the prevalence of lamb mortality can behigh up to93%. The disease can cause huge economic loss for ovine husbandry and threaten the health ofhuman.DNA polymerase、VETF-S and VETF-L genes which the virus needed to copy were selected as thetargeted gene. The primers of the targeted gene were designed, then the targeted gene were obtained by PCR,which were connected with Check2vector digested by PmeI and NotI. Check2-targeted gene vectors wereconstructed．In accordance with RNAi sequence design principles in the public Web site, eleven RNAi targetsequences were designed, then the target sequences of Oligo DNA were synthesized and annealed todouble-stranded DNA, which were connected with pll3.7vector digested by XhoI and HpaI. Pll3.7-shRNAvectors were constructed.293T cells were co-transfected by pll3.7-shRNA vectors and Check2-targeted genevector. The interference rate was assayed by the Dual-GloTMLuciferase Assay System.And the bestinterference rate was91.0%. Three cell lines for inhibiting Orf virus were established, which can expressP-D-3、P-L-2、P-S-2siRNA. The inhibiting rate of the ORFV replication was evaluated by The virus′titer、theexpression of proteins. Theses results provided a valid basis for preventing and treating the ORFV and theproduction of disease-resistant transgenic livestock.